Aquaporin Channel Physiology and Drug Discovery Laboratory
Aquaporins (AQPs) are membrane channels that allow water and solute movement across specialized cells and tissues. Our research focuses on AQPs in the mammalian nervous system that enable essential fluid homeostasis, and a fly homolog, Big Brain, required in early nervous system development. Our interdisciplinary research team uses molecular biology, electrophysiology, cell culture, and imaging to assess the links between AQP three-dimensional protein structure and channel function, and to dissect the sophisticated roles of these channels as water pores and ion channels. Site-directed mutagenesis and voltage clamp of cloned AQPs expressed in frog oocytes are being used to define the barriers and gates in AQP permeation pathways. Little is known regarding the pharmacology of AQPs. Using clues from clinical literature and herbal lore, theoretical modeling, and experimental testing, our lab has discovered lead compound blockers for AQP1 and AQP4 with potential significance in cerebral edema, hydrocephaly, and glaucoma. Opportunities to contribute to ongoing work involve characterization of mechanisms of AQP and BIB channel regulation, analyses of AQPs in signaling complexes, drug discovery of new blocking compounds, definition of the molecular binding pockets, and exploration of these novel pharmacological tools to probe physiological significance of AQPs in health and disease.
Molecular basis of gating in Aquaporin-1 channels
Aquaporin-1 (AQP1) mediates the secretion of fluid in the eye and brain by a mechanism that we have proposed involves parallel pathways through the same molecule: pores for water in the four individual subunits that comprise the channel, and a gated pore for ions in the center of the tetramer that is opened by intracellular cGMP. We will study the dual ion channel and water channel properties of cloned human AQP1 heterologously expressed in the Xenopus oocyte (a popular system for the electrophysiological analyses of cloned channels and transporters). Techniques to be learned include: Molecular methods of DNA amplification, linearization and purification, in vitro synthesis of RNA; and cellular methods for protein expression and the quantitative analyses of water fluxes (by video microscopy) and measurement of ion currents (by two electrode voltage clamp). In addition to the wild type AQP1 constructs, a large array of site-directed mutant channels are available to probe the molecular basis for the physiological functions of the AQP1 channel. The project will focus on one of a number of candidate regulatory domains identified by sequence analysis in the amino and carboxyl terminal domains, but whose functional roles remain open for discovery.
Regulation of the Big Brain channel by intracellular signalling cascades
In early development of the fly nervous system, the neurogenic protein “Big Brain” (BIB) serves an essential role in the lateral inhibition process of cell-cell signaling that determines cell fate. When BIB is defective, the fly brain overgrows, due to an over-production of neuroblasts at the expense of epidermoblasts. We have found that the signaling function of BIB involves a cationic conductance regulated by tyrosine kinases, but the details of the cascade that is induced in oocytes (or in flies) remains unknown. The project will use pharmacological dissection of intracellular second messenger pathways to define the signaling pathway that controls ion channel activation of cloned Drosophila BIB, heterologously expressed in Xenopus oocytes. Techniques to be learned include: Molecular methods of DNA amplification, linearization and purification, in vitro synthesis of RNA; and cellular methods for protein expression and the quantitative measurement of ion currents by two electrode voltage clamp. In addition to the wild type BIB constructs, a large array of site-directed mutant channels lacking consensus tyrosine phosphorylation sites are available to probe the molecular basis for the ion channel activation. The endogenous insulin-related growth factor receptor present in oocytes is used to drive the tyrosine kinase pathways; selective pharmacological blockers for kinases and phosphatases are used to identify necessary signaling elements in the hierarchical regulatory pathway in the oocyte system.
Drug discovery for mammalian Aquaporins
Aquaporins are important factors in potentially life-threatening brain edemas induced by stroke or injury, lung edemas, and in conditions that compromise quality of life, such as glaucoma which can lead to blindness. Despite their medical importance, very little is known about agents that could serve as pharmacological blockers of AQPs. Using the Xenopus oocyte system for expressing cloned mammalian aquaporin channels (AQP1, 4, 5 and 6), the project will explore sources of selected clinical agents and natural products as possible sources for novel blockers of AQPs. The assay for blocking activity uses video-camera imaging of the cross-sectional areas of oocytes expressing wild type AQPs as a function of time after transfer into low-salt solution, and computer-assisted analysis to quantify net water flux. Techniques to be learned include: Molecular methods of DNA amplification, linearization and purification, in vitro synthesis of RNA; and cellular methods for protein expression and the quantitative measurement of water flux (by swelling rate in hypotonic saline). Compounds are selected based on clues compiled from the clinical literature database and from traditional herbal knowledge, as well as from existing chemical libraries. For this project, approximately 50 compounds will be quickly tested in a simple rapid screen, and a small set (2-3) of the most promising candidates will be chosen for in-depth characterization of potency, selectivity and dose-dependence.
Lab Head: Andrea Yool
Postdoctoral Researcher: Ewan Campbell
PhD candidate: Victor Pei
Honour candidates: Claire Hoban